PROJECT SUMMARY Our work is designed to provide new insights into understanding of signaling mechanisms responsible for the developmental control of organ growth. Our preliminary data have implicated Capicua (Cic) as a target of growth- controlling signaling pathways and suggest that Cic integrates several upstream signals to control organ size. Cic is a transcriptional repressor protein that is regulated by the receptor tyrosine kinase (RTK)-extracellular signal regulated kinase (ERK) pathway, which is one of the key systems involved in organ size control and tissue patterning in humans and model experimental animals. Our preliminary studies have identified a kinase Minibrain (Mnb) as a novel Cic regulator which acts in parallel to RTK/ERK signaling. ERK signaling components, Mnb (human DYRK1A), and Cic are highly conserved proteins, and alterations in their levels in humans result in several diseases, such as Rasopathies, neurodegenerative disorders, and cancer. DYRK1A has been actively investigated as one of the causative factors in Down syndrome, and is thought to be important for proper development and growth of the central nervous system. An exciting hypothesis that we pursue in this application is that signals from ERK and Mnb converge on Cic to regulate its activity as a growth suppressor. Given that human pathologies can result from quantitative changes in ERK and DYRK1A signaling, it is essential to apply quantitative approaches in order to understand the underlying mechanisms. Drosophila offers a unique opportunity to carry out such research in vivo. We propose to use this powerful experimental platform to carry out a quantitative and systems-level analysis of developmental signals controlling growth. We will use state-of- the-art proteomic methods, such as affinity purification mass spectrometry (AP-MS), to identify the binding partners of Cic in the embryo and analyze how upstream signals alter these interactions, which in turn leads to changes in Cic activity. We will then apply quantitative imaging assays to systematically test the identified interacting partners for their involvement in three molecular outcomes: Cic degradation, nuclear export, and inhibition of transcriptional repressor activity. Furthermore, we will investigate how Cic integrates signals from the ERK and Mnb kinases by identifying and functionally validating the phosphorylation sites targeted by these two kinases, and by testing the relative contributions of these kinases to Cic regulation and growth control in four independent in vivo assays. In summary, our multi-level experimental plan is designed to provide insights into the molecular mechanisms of RTK/ERK signal interpretation by Cic, as well as to determine how these mechanisms intersect with the input from Mnb, a novel Cic regulator. In the long term, this work will advance our understanding of the complex regulatory relationships between the pathways involved in organ size control, and may suggest new targets for developing ERK, Cic, and DYRK1A-related therapies.